1. Field of the Invention
The present invention generally relates to cell disruption (lysing) methods and equipment. More particularly, this invention relates to a lysing device and method that utilize a vibrating micromachined tube through which biological cells, spores or other cellular matter flows to produce or promote the cell lysing process.
2. Description of the Related Art
Cell disruption, or lysis, involves releasing the biological molecules within a cell by physically or chemically rupturing the cell wall. Lysis of biological cells and spores is used in a variety of biological research and development activities, including the analysis of DNA, the isolation of RNA, the extraction of hemoglobin from red blood cells, the separation of cellular parasites, the detection of pathogens, enzyme concentration, vaccine production, and cancer cell research. After lysing, filtration may be performed to remove small fragments of the cell walls if total cell disruption has occurred, immediately followed by analysis of the released biological molecules and cellular components (lysate) to avoid denaturation of the released materials. Various chemical and biological analysis techniques may be performed on the lysed material, such as chromatography and electrophoresis. Electrophoresis can be used to separate different elements (fractions) of a blood sample into individual components, such as proteins to evaluate, diagnose and monitor a variety of diseases and conditions. For example, in a process known as serum protein electrophoresis (SPEP), a lysed blood sample is placed in or on a special medium (e.g., a gel), and an electric current is applied to the gel to cause the protein particles to move through the gel according to the strengths of their electrical charges. These moving proteins form bands or zones that can be detected and identified for use in diagnosing specific diseases.
Cell disruption is generally accomplished by either mechanical or chemical techniques. The type of tissues or cells to be lysed often necessitates certain methods or combinations of methods. Chemical techniques use enzymes or detergents to dissolve the cell walls, and are usually followed by sonication, homogenization, vigorous pipetting or vortexing in a lysis buffer. Mechanical lysing is more widely practiced, and may be accomplished with a mortar and pestle, bead mill, press, blender, grinder, high pressure (e.g., up to 1500 bar), nozzle, or a probe or membrane operated at ultrasonic frequencies. Micromachined devices that use sharp comers or high pressure nozzles to rupture cell walls have also been developed to perform mechanical lysing. As used herein, micromachining is a technique for forming very small elements by bulk etching a substrate (e.g., a silicon wafer), or by surface thin-film etching, the latter of which generally involves depositing a thin film (e.g., polysilicon or metal) on a sacrificial layer (e.g., oxide layer) on a substrate surface and then selectively removing portions of the sacrificial layer to free the deposited thin film.
Ultrasonic lysing operates on the basis of generating intense sonic pressure waves in a liquid medium in which the cellular material of interest is suspended. The pressure waves are transferred to the medium with a probe or membrane, and cause the formation of microbubbles that grow and collapse violently, generating shock waves that, if at a sufficiently high energy level, can break cell membranes. One type of known ultrasonic device employs a piezoelectric generator made of lead zirconate titanate crystals to induce vibration in a titanium metal horn or probe tuned to resonate at 15 to 25 kHz. Glass beads may be added to the liquid medium to promote lysis of certain materials.
While lysing techniques and devices of the type describe above have been successfully employed, there is a continuing effort to develop improved devices for performing lysing, as well as to perform pre- and post-lysing steps, such as mixing, agitating or homogenizing a fluid after lysis, or before, during or after other processing performed on lysate, such as incubation and mixing.